Device and method for detecting bioelectric signals from electrophysiologically active regions in spheroids

ABSTRACT

Disclosed is a device for detecting bioelectric signals from spheroids comprising a measuring chamber having a measuring chamber wall which encloses a volume, which is open at least at one side, is composed of an electrically non-conducting material, and has, in at least one measuring region, an inner cross section, which corresponds as far as possible to the largest cross section of a spheroid, comprising at least a number of electrodes which are disposed in a common plane inside said measuring chamber wall and each electrode has a freely accessible electrode surface which is oriented towards the measuring region, and comprising an impedance measuring arrangement which is connected to the electrodes. The device and the method can be used to test substances in 3D biological in-vitro (three-dimensional) cell aggregates.

TECHNICAL FIELD

[0001] The present invention relates to a device and a method fordetecting bioeletric signals from electrophysiologically active regionsin spheroids. In particular, it is described how the effect ofpharmaceutical preferably neuropharmacological or neurotoxic substancescan be detected without damaging the spheroids so that the spheroidscontinue to be at disposal for further study possibilities.

STATE OF THE ART

[0002] In order to be able to routinely determine the effect ofsubstances, for example pharmacological substances, on living systems,in recent years biosensors have been developed, which are based onliving cells, see Bousse, L., “Whole Cell Biosensors”, Sensors andActuators, volume 34, pp., 270-275 (1996). Such type biosensors that arebased on biological cells are primarily provided with mono-layer cellcultures as a biological detection system, but substance-caused complexcell/cell or cell/matrix interactions can often not be determined withthe desired precision and reliability. Furthermore, the effect ofneuropharmaceuticals or environmental toxins indeed leads to thesecomplex cell/cell interactions in the central nervous system, which needto be ascertained in order to obtain further insight into thebiochemical reaction chain of such substances on biological cellmaterial. Finally, the biosensors based on mono-layer cell cultures havethe drawback that the measuring results obtained using them only providelimited information about the actual reaction capabilities of thebiological cells, for example to selective application of a substance,because the mono-layer cell cultures do not exist in this from in livingnature.

[0003] In order to avoid this drawback, biological models approximatingan in vivo situation with regard to the intercellular as well asintracellular interactions as closely as possible must be resorted towhen studying such type substances. Three-dimensional cell systemsreflect an in vivo situation substantially better than single cells ormono-layer cell cultures. Therefore, it is necessary to usethree-dimensional cell systems to test substances which are intended forinfluencing cell/cell interactions.

[0004] In order to test the neuropharmacological or neurotoxic effect ofsubstances, for example beyond animal models, bioelectric signals aredetermined in a prior art manner from the ex vivo tissue sections withthe aid of glass micro-electrodes or needle electrodes. Planar electrodearrangements, so-called multi-electrode arrays are utilized to recordthe signal courses using multi-channel derivations. However, ex vivotissue sections must be prepared in a very complicated manner fromanimal models, cannot be standardized and are limited to the existentanimals models. Moreover, as ex vivo tissue sections degenerate rapidly,tissue sections are not suited for long-term testing. Long-term testing,however, is of extreme relevance for testing neuropharmaceuticals orenvironmental toxins and their influence on biological tissue.

[0005] An interesting research object for the preceding problem areso-called spheroids, which may be considered as bead-shaped cellaggregates. From literature are known, for example, research in retinagenesis and retina regeneration in which such type regeneratedbead-shaped cell aggregates, so-called retino-spheroids, are obtainedunder constant conditions (see Moscona, A. A, “Development ofHeterotypic Combination of Dissociated Embryonic Chick Cells”, Proc.Soc. Exp. Bio. Med 292, pp. 410-416 (1956); Vollmer, G. Layer, P. G.,Gierer, A.: “Reaggreation of Embryonic Chick Retina Cells: PigmentEpithelial Cells Induce a High Order of Stratification”, Neurosci. Lett.48, pp. 191-196 (1984)). These regenerated bead-shaped cell aggregatesare reaggregated by suited cultivation of dissociated cells fromembryonic retinae.

[0006] DE 199 46 458.8 describes a device and a method forcharacterizing spheroids by means of impedance spectroscopy. Theinfluence of substances on the proliferation, morphology and membraneproperties of the in vitro tissue, i.e. outside the living organism, canbe determined with this device and method. Locally resolved informationfrom inside the spheroid can, however, not be obtained with thisprior-art method. Moreover, information about the intracellular electricpotentials in the form of so-called bioelectric signals, from which theeffect of pharmaceutical substances, in particular neuro-pharmaceuticalor neurotoxic substances can be determined, cannot be obtained with thedevice described in the preceding printed publication.

SUMMARY OF THE INVENTION

[0007] The object of the present invention is to provide a device and amethod for detecting bioelectric signals from spheroids in such a mannerthat it is possible to determine the neurotoxic and neuropharmacologicaleffect of substances on biological tissue by way of in-vitro study asclose as possible to the in-vivo situation with regard to intercellularand intracellular interactions.

[0008] The solution of the object on which the present invention isbased is set forth in claim 1. The subject matter of claim 14 is aninventive method. Features that advantageously develop the inventiveidea are the subject matter of the subclaims and are given in thedescription of the invention with reference to preferred embodiments.

[0009] A key element of the present invention is that the device fordetecting bioelectric signals from spheroids comprises the followingcomponents:

[0010] a measuring chamber having a measuring chamber wall, whichencloses a volume that is open at least on one side, is made ofnon-electroconductive material and has an inner cross section at leastin one measuring region, which corresponds maximally to the largestcross section of a spheroid.

[0011] The measuring chamber is preferably designed as a capillary withcapillary walls as well as a capillary bottom, which define themeasuring region of the spheroid. The size of the cross section of themeasuring region enclosed by the capillary walls is selected in such amanner that the spheroid is in mechanical contact with the measuringchamber respectively capillary wall along the spheroid's biggestcircumferential edge in such a manner that the spheroid assumes a fixedas possible spatial position inside the measuring region, which is ofgreat advantage for further measurement of the spheroid. In order tofurther improve the positioning of the spheroid inside the measuringchamber, respectively the capillary, in a preferred embodiment, thedevice is connected in the capillary bottom to a partial vacuum conduitto affix the spheroid inside the measuring region by means of suction.

[0012] at least a number of electrodes which are disposed in a commonplane inside the wall of the measuring chamber, the electrodes eachhaving one freely accessible electrode surface oriented towards themeasuring region. The electrodes are preferably disposed in that planewithin the wall of the measuring chamber, in which the spheroid comes incontact with the measuring chamber wall with the edge of its greatestcircumference. The requirement that the electrodes are disposed in aplane is not necessarily to be understood as mathematically exact, i.e.in the sense of along an imaginary line running around the inner wall ofthe measuring chamber. The electrodes should be disposed, at least withthe surfaces oriented towards the measuring region, along the region ofcontact between the spheroid and the wall of the measuring chamber insuch a manner that the impedance distribution can be detected locallyresolved in the cutting plane inside the spheroid predetermined by theelectrode configuration with an impedance measuring arrangement that isconnected to the individual electrodes. For this purpose, an electriccurrent is induced inside the spheroid via the single electrodes and thediminishing electric voltage over the spheroid is measured. Theimpedance is formed from the current and the voltage. In order toconduct so-called impedance imaging, the frequency of the currentinduced in the spheroid is varied over a continuous frequency range, andthe impedance yielded as a function of the frequency is recorded. Thus,with the aid of such an impedance imaging system, the tissue parameterscan be determined locally resolved inside the cutting plane from therecorded impedance distribution at different frequencies. In thismanner, information is gained about the internal structure of thespheroid inside the cutting plane. For instance, so-calledelectrophysiologically active regions, which may differ in impedancebehavior from the other not organized regions inside the spheroid, aredistinguished by a subunit with a compact consistency. Especially theseelectrophysiologically active regions are of particular interest indiscovering how certain substances influence biological cells, becauseit is in these regions that scientifically detectable and evaluatablesignals are generated as a sort of cell response to the substance'seffect on the respective cell. Electrophysiologically active regionsinside the spheroid possess a bioelectrical activity which influencesthe behavior of the electrical surface potential of the entire spheroid.When there is a change in the bioelectrical activity of theelectrophysiological active regions, for example, due to the effect of acertain substance on the spheroid and, therefore, simultaneously on theelectrophysiologically active regions, this directly influences thesurface potential of the spheroid. Preferably with the aid of apotential determining system, which is connected to the electrodesdisposed around the spheroid, it is possible to detect the surfacepotentials along the cutting plane through the spheroid and to obtaininformation about the bioelectrical activity of theelectrophysiologically active regions inside the cutting plane.

[0013] For both impedance measurement and detection of the surfacepotentials, the free electrode surfaces do not necessarily have to be indirect contact with the surface of the spheroid.

[0014] But rather a culture fluid, for example representing a nutrientinside which the spheroid is generated, introduced into the measuringchamber also acts as an electrically conducting medium through which anelectrical contact can be produced between the electrodes and thesurface of the spheroid.

[0015] In a simple embodiment, the free electrode surfaces-connect flushwith the inner wall of the measuring chamber in such a manner that adirect contact between the electrode surfaces and the spheroid prevails.

[0016] In an alternative embodiment, the electrodes are located in sucha manner inside the so-called connecting chambers, which open on oneside into the measuring chamber, that the free electrode surfaces areset back from the inner wall of the measuring chamber. The advantage ofthis is first that the electrodes are easier to exchange respectivelyreplace. Moreover, with suited design and arrangement of the connectingchamber, for example, tapering conically towards the measuring chamber,larger free electrode surfaces can be utilized. With regard to a smallas possible phase limit impedance, the use of as large as possibleelectrode surfaces is desirable, which can be realized by correspondingspaced placement of the inner wall of the measuring chamber inside theconically designed connecting chambers. As already mentioned in thepreceding, the culture fluid, which is introduced into the measuringchamber together with the spheroid, acts as an electrical contact mediumbetween the electrodes and the spheroid surface.

[0017] With regard, in particular, to studying spheroids in industrialamounts to test how new pharmacological substances act, semiconductormaterials are suited for-setting up the device described in thepreceding. A multiplicity of array-like arranged measuring chambers,which are adapted in shape and size to studying spheroids and thuspermit statistical evaluation due to the great number of examinedspheroids, can be realized with the aid of semiconductor technology. Aconcrete embodiment of this is described in more detail further on withreference to the figures.

[0018] With the aid of the preceding device, the spheroids can bestudied for their bioelectrical activity without destroying them, tothen return them safely to a culture medium for further observation.Thus, one and the same spheroid can be measured several times atintervals in order to be able to determine possible signs ofsubstance-caused degradation. In this manner, conclusions can be drawnstatistically about how substances act following evaluation of amultiplicity of such spheroids which are additionally exposed to acertain substance inside a culture medium.

[0019] The invented method for detecting bioelectric signals fromspheroids is distinguished in particular by the combination of thefollowing method steps: provision of a device of the type described inthe preceding, placement and positioning of a spheroid inside themeasuring chamber, and conducting an impedance measurement according tothe impedance imaging method for locally resolved determination ofelectrophysiologically active regions in the spheroid. In order to beable to determine any bioelectrical activity, an additional surfacepotential determination is conducted along the cutting planepredetermined by the configuration of the electrodes.

[0020] With the aid of the invented method, the morphology of themulti-cellular spheroids can be determined locally resolved in anon-invasive manner and, moreover, the excitation courses of theelectrophysiologically active regions can be precisely determined. Themethod permits, in particular, to be able to non-invasively detect theeffect of substances respectively drugs on 3D in vitro models of thecentral nervous system. The device, in the sense of a biosensor systemdescribed in the preceding, permits the realization of long-term studiesof neurotoxic and neuropharmacological effects of substances. Thespheroid utilized as a biological detection element is only positionedin the measuring chamber for a short period during impedance measurementand potential determination and can, independent of the measuringarrangement, be cultivated under physiological conditions. Adhesion ofthe spheroid is largely prevented by the presence of the culture fluidinside the measuring chamber and undesired cell/material interactionsare minimized. Depending on the question to be resolved, spheroids or 3Dbiological detecting elements can be generated with different types ofcells in different positions for the biosensor system.

BRIEF DESCRIPTION OF THE DRAWING

[0021] The present invention is made more apparent in the following,without the intention of limiting the scope or spirit of the overallinventive idea, using preferred embodiments with reference to theaccompanying drawings. Shown is in:

[0022]FIG. 1 a schematic flow chart representation of carrying out themethod of analysis,

[0023]FIGS. 2a,b representation of a measuring chamber with a spheroid,

[0024]FIGS. 3a,b,c representations of cross sections of a spheroid andan-image of the potential,

[0025]FIG. 4 a cross section of an array-shaped measuring arrangement insemiconductor technology,

[0026]FIG. 5 measuring arrangement

[0027]FIG. 6 a cross section of a measuring chamber and

[0028]FIG. 7 a cross section of an alternative measuring chamber.

WAYS TO CARRY OUT THE INVENTION, COMMERCIAL APPLICABILITY

[0029] The invented method is explained with reference to FIG. 1 using astudy of reaggregated retino-spheroids as an example:

[0030] Under micro-gravitation conditions in a bioreactor 1, dissociatedembryonic cells of the central nervous system are reaggregated tobead-shaped neuronal reaggregation cultures, the so-calledretinospheroids. With the addition suited growth factors and/or suitedgenetic manipulations, it is achieved that electrophysiologically activecell regions form evenly distributed in the spheroid. Thus with highprobability, at least one electrophysiologically active region islocated in a random cutting plane running through the center of thespheroid.

[0031] In order to test the effect of a substance on the spheroid, atleast one spheroid 2 has to be isolated from the bioreactor 1 and placedin the measuring chamber of the biosensor system 3 to test it there asan in vitro model. In the bioreactor 1 as well as in the measuringchamber of the biosensor 3, the spheroid 2 is located in a culturefluid, respectively in an analyt, so that the spheroid is replaceable asdesired without impairment between the bioreactor and the measuringchamber.

[0032] By means of multi-frequency impedance imaging 4, the position andthe extension of the various cell regions is determined in a crosssection plane predetermined by the electrode configuration inside themeasuring chamber. And then the bioelectrical activity of the individualcell regions in the cutting plane is detected by means of the electricalsource-imaging 5. Systems and algorithms for the impedance imaging andthe electric source imaging are fundamentally known from medicaltomography, see Webster, J. G., “Electrical Impedance Tomography”, AdamHilger, Bristol (1990).

[0033] Changes in the electrophsiological activity of certain regions inthe spheroid, correlation of the electrophysiological activity ofdifferent regions and the change in the tissue parameters serve asparameters 6 for the effect of substances on the in vitro tissue model.

[0034] To conduct the impedance imaging and the potential determination,an electrophysiologically active spheroid 2 according to FIG. 2 ispositioned in the desired culture stage in a measuring chamber 7.Depending on the question to be resolved, such as for example long-termstudies or dynamic excitation, the to-be-tested substance is added tothe culture fluid in the bioreactor or to the culture fluid in themeasuring chamber 7. The measuring chamber 7 is preferably formed by acapillary 8, which is designed cylindrical in the positioning region andits wall 9 is made of an electrically insulated material. In thepositioning region of the capillary 8, a multiplicity of electrodes 10are disposed in the capillary wall 9 in at least one plane perpendicularto the longitudinal axis. As the electrodes 10 with their free electrodesurfaces in the preferred embodiment shown in FIGS. 2a,b are placedflush with the inside wall of the measuring chamber, they come incontact with the spheroid (2) placed inside the measuring chamber in acutting plane along the spheroid's greatest circumference (FIG. 2b). Theelectrodes are electrically contacted singly from the outside fortriggering (not depicted).

[0035] This arrangement is used both for locally resolved determinationof the passive electrical properties of the in-vitro tissue and fordetermination of the spatial and temporal course of theelectrophysiological excitation.

[0036] In order to determine the impedance distribution in the cuttingplane of the spheroid in which the electrodes lie, the electrodes areconnected in a suited manner with an impedance imaging system. Thetissue parameters are determined locally resolved from the impedancedistributions at different frequencies. FIG. 3a shows an actual sectionthrough a spheroid with the usually not further organized regions 11,organized subregions, the so-called electrophysiologically activeregions 12 and inner fiber layers 13. FIG. 3b shows a section imagedetermined by means of impedance imaging, which really corresponds tothe section image shown in FIG. 3a. The bioelectrical activity ofcertain regions is determined from the surface potentials, which aredetermined with the electrodes, and from the impedance distribution, seediagrammatic representation in FIG. 3c. In the determination of thesurface potentials, the electrodes of the measuring capillaries areconnected to a measurement system. Marked evaluatable measured signalsare, in particular, the voltages peaks perceivable on the surface of thespheroid (see diagrammatic representation) and their temporal sequence(Δt₁, Δt₂). In particular, these measured values are the ones that areprovenly influenced by the presence of certain substances, which allowsdrawing conclusions about the effect of certain substance on biologicalmaterial.

[0037]FIG. 4 shows a measuring chamber arrangement in which amultiplicity of single measuring chambers 7 is disposed in an arraystructure on a planar substrate 14. In order to produce such a measuringchamber arrangement, a silicon nitride layer 15 with a thickness ofapproximately 1 μm is deposited on a silicon substrate 14. The siliconnitride layer 15 is exposed from the underside as membranes.Furthermore, microholes 16 with a diameter of 20 μm are produced in themembrane 15 by means of dry etching. Finally, a phototresist 17 with athickness of 40 μm is applied. Cylindrically shaped measuring chambers 7(diameter of 150 μm) are etched free into the photoresist 17 concentricto the microholes. A metal layer with a thickness of 10 μm is depositedonto the photoresist 17 and structured in such a manner that themeasuring chambers 7 are surrounded by eight electrodes 10 disposedevenly spaced in a circle. Finally another photoresist layer 17 (50 μm)is deposited and structured.

[0038] In order not to damage the spheroids when placing them into theindividual measuring chambers 7, the edges of the measuring chambers 7are rounded off. In order to be able to apply a partial vacuum 18 forpositioning the spheroids, the finished microstructure is glued onto aplate with a borehole and tube connection. To conduct a measurement, theentire region of the measuring chamber 7 is filled with culture fluid 19to prevent adhesion effects between the individual spheroids and themeasuring chamber wall.

[0039] The electrodes 10 placed in the measuring chamber, according toFIG. 5, are connected with an impedance measuring system 21 and apotential determination system 22 via a multiplexer 20. The measureddata are transmitted to a data collection and data analysis unit 23,which also controls the multiplexer 20.

[0040] According to the preferred embodiment in FIG. 6 showing a crosssection of a measuring chamber 7, connecting chambers 24, which end inthe measuring chamber, are disposed in a star-shaped manner runningconically into the measuring chamber 7. In this way, the size of themetal electrodes 10, which are each contacted by strip conductors 10*,can be selected independent of the size of the measuring chamber, andthe phase limit impedance of the electrode 10 can be reduced by largerelectrode surfaces.

[0041] In another preferred embodiment according to FIG. 7, in order torealize impedance measurements, one electrode 10′ for supplying currentand one electrode 10″ for determining the potential are disposed in eachchannel in a four-electrode configuration.

List Of Reference Numbers

[0042]1 bioreactor

[0043]2 spheroid

[0044]3 biosensor, measuring arrangement

[0045]4 impedance measuring arrangement

[0046]5 potential determining arrangement

[0047]6 evaluation parameter

[0048]7 measuring chamber

[0049]8 capillary

[0050]9 measuring chamber wall

[0051]10 electrode

[0052]10* strip conductor

[0053]10′ current supply electrode

[0054]10″ potential determining electrode

[0055]11 not organized region

[0056]12 organized region, electrophysiological region

[0057]13 inner fiber layer

[0058]14 substrate

[0059]15 membrane

[0060]16 microhole

[0061]17 photoresist

[0062]18 vacuum connection

[0063]19 culture fluid

[0064]20 multiplexer

[0065]21 impedance system

[0066]22 potential determining system

[0067]23 data collection and analysis system

[0068]24 connecting chamber

1. A device for detecting bioelectric signals from spheroids comprisinga measuring chamber having a measuring chamber wall which encloses avolume which is open at least at one side, is composed of anelectrically non-electro conducting material and has, in at least onemeasuring region, an inner cross section which corresponds as far aspossible to the largest cross section of a spheroid, at least a numberof electrodes which are disposed in a common plane inside said measuringchamber wall and each said electrode has a freely accessible electrodesurface which is oriented towards said measuring region, and animpedance measuring arrangement which is connected to said electrodes.2. The device according to claim 1, wherein a potential determiningsystem is connected to said electrodes.
 3. The device according to claim1 or, wherein said measuring chamber is designed as a cylindricalcapillary, and said at least a number of said electrodes is disposed inone plane orthogonally to the length of said capillary.
 4. The deviceaccording to claim 1, wherein said freely accessible electrode surfacesof said electrodes are designed flush with said measuring chamber innerwall.
 5. The device according to claim 1, wherein provided in saidmeasuring chamber wall is a number of connecting chambers which areopenly connected with said measuring region and are disposed in a commonplane evenly distributed around said measuring region in circumferentialdirection, and inside each of said connecting chamber an electrode isplaced, whose said freely accessible electrode surface oriented towardssaid measuring region is spaced at a distance from said measuringchamber inner wall.
 6. The device according to claim 1, wherein saidmeasuring chamber can be filled with an electrically conducting liquid.7. The device according to claim 5, wherein a second electrode isarranged inside each of said connecting chambers and being connected tosaid potential determining system.
 8. The device according to claim 1,wherein a partial vacuum conduit is connected to said measuring chamber.9. The device according to claim 1, wherein said measuring chamber isdesigned pot-shaped, and a partial vacuum conduit is provided at thebottom of said pot for positioning and attaching a spheroid placed insaid pot-shaped measuring chamber by means of a partial vacuum.
 10. Thedevice according to claim 1, wherein said impedance measuringarrangement and said potential determining system are connected to saidelectrodes via a multiplexer.
 11. The device according to claim 1,wherein a data collecting and data evaluation unit is connected to saidimpedance measuring arrangement as well as to said potential determiningsystem.
 12. The device according to claim 1, wherein a multiplicity ofmeasuring chamber is arranged in an array-like manner and is designed inplanar semiconductor substrate technology.
 13. The device according toclaim 1, wherein said electrodes are disposed evenly distributed incircumferential direction of said measuring chamber wall.
 14. A methodfor detecting bioelectric signals from spheroids with the followingsteps: providing of a device according to claim 1, placing andpositioning of a spheroid inside said measuring chamber, conducting animpedance measurement according to the impedance-imaging method forlocally resolved determination of electrophysiologically active regionsin said spheroids.
 15. The method according to claim 14, wherein asurface potential determination is conducted for detecting saidbioelectrical activity.
 16. The method according to claim 14, whereinsaid impedance measurement is conducted at different triggeringfrequencies to obtain an impedance spectrum.
 17. Use of said deviceaccording to claim 1 to study the effect of substances on spheroids. 18.Use according to claim 17, wherein said substances are pharmaceuticalsubstances, in particular, neuropharmacological or neurotoxicsubstances.
 19. Use according to claim 17, wherein in a first step, aspheroid to which a substance has been applied is removed from a culturemedium and placed in said measuring chamber of said device, in asubsequent step, said impedance measurement and/or said potentialdetermination are carried out non-invasively on said spheroid, and in afinal step, said spheroid is returned unharmed to said culture medium.